Method for obtaining homozygous mutation of seamless modification

A homozygous, seamless technology, applied in the field of molecular biology, can solve the problems of low efficiency of precise and seamless gene modification, troublesome construction of plasmids, lack of screening system, etc., to save manpower, material resources and time, facilitate disease models, and select the range wide range of effects

Inactive Publication Date: 2018-03-23
UNIVERSITY OF MACAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Double-strand breaks are necessary for homologous recombination to obtain genetic mutations. Of course, this also stimulates another, more common endogenous DNA repair pathway - non-homologous end joining (NHEJ), which will be precise and seamless. Genetic Modification Creates Barriers
Synthetic ssODNs can be conveniently ordered from companies, but it lacks a screening system, making precise and seamles

Method used

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  • Method for obtaining homozygous mutation of seamless modification
  • Method for obtaining homozygous mutation of seamless modification
  • Method for obtaining homozygous mutation of seamless modification

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0033] Example 1 CRISPR / Cas9 combined with piggyBac transposon carrying puro / TK resistance screening to efficiently obtain heterozygous mutations

[0034] experimental method

[0035] 1. Cell culture and transfection

[0036] Plant iPS cells on cell culture plates pre-coated with Matrigel (BD Biosciences), and use mTeSR TM 1(Stemcell Technologies) at 37℃, 5% CO 2 In the cell culture incubator. Before transfection with Lipofectamine, press iPS cells 4×10 per well 5 ~5×10 5 The density was passaged on a six-well plate coated with Matrigel, using mTeSR containing 10μM ROCKinhibitor (Sigma) TM 1 Incubate overnight. On the second day after passaging, iPS cells were co-transfected with 6μg pCas9_NeoR, 3μg sgRNAplasmid, and 50nMssODN (90bpssODN). The other well of cells were transfected with 2μg pmaxGFP as a control of transfection efficiency. The transfection procedure was strictly in accordance with Lipofectamine TM 3000 official operating instructions. In all transfection experimen...

Example Embodiment

[0056] Example 2 CRISPR / Cas9 combined with piggyBac transposon containing puro / TK and hygR / TK resistance screening to obtain homozygous mutations efficiently

[0057] experimental method

[0058] The experimental method is the same as that used in Example 1.

[0059] 1. Cell culture and transfection

[0060] The same as in Example 1.

[0061] 2. iPS single cell clone culture

[0062] The same as in Example 1.

[0063] 3. Construction of Cas9 and PB-based plasmids

[0064] The same as in Example 1.

[0065] 4. Obtain homozygous mutations

[0066] The following still takes PSEN1ΔI83M84 as an example to illustrate the process of obtaining seamless homozygous mutations using CRISPR / Cas9 and the new piggyBac transposon system, such as Figure 2A Shown:

[0067] (a) First, determine that the site to be mutated is on exon 4 of PSEN1;

[0068] (b) Use Cas9 to cut at the target site to generate DSB;

[0069] (c) Construction of piggyBac transposons (SEQ ID No: 7 and SEQ ID No: 8) carrying puro / TK and Hy...

Example Embodiment

[0076] Example 3 Experimental method for cells carrying AD-related gene mutations to obtain gene mutation-dependent phenotypic changes

[0077] 1. Differentiation of cortical neurons

[0078] Human iPS cells differentiate into neural stem cells, and the cultured iPS cells are scraped into small clonal clusters with a size of 2.5-3.0×10 4 Cells / cm 2 It was cultured on a 6-well plate pre-coated with matrigel (BD Biosciences) with PSC Neural Induction Medium (Life Technologies) (Life Technologies) and Y23632 (sigma). The medium was changed every two days for continuous culture for 7 days. After 7 days, use Accutase (Sigma) to digest single cells to 1.0-1.2×10 5 Cells / cm 2 The density is expanded and cultivated.

[0079] For the differentiation of cortical neurons, on a cell culture plate pre-coated with poly-L-ornithine / laminin, neural stem cells are divided into 2×10 5 Cells / cm 2 The density of N2B27 medium (DMEM-F12: Neural Basal medium 1:1, 2% B27, 1% N2, 1% non-essential aminoacids)...

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Abstract

The invention relates to a method for obtaining homozygous mutation of seamless modification. The method comprises the following steps: (1) cutting a locus needing mutation in a cell genome to generate double-chain breaking by using a gene editing system; (2) respectively integrating two kinds of transposons, which contain a target mutation sequence and different resistance, to enter a pair of alleles of the cell genome through homologous recombination; (3) screening a cell clone of the alleles which integrate the transposons, and then cutting off the transposons; and (4) causing death to thecell clone in which the transposons are residually left to obtain the cell clone which contains the homozygous mutation of the seamless modification. The method can be used for obviously improving theprobability of obtaining the homozygous mutation, manpower, material resources and time are saved, and the application range is wide.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for obtaining seamlessly modified homozygous mutations. Background technique [0002] Compared with animal models, cell models derived from human iPS have a series of advantages, such as being able to be purified into a specific cell type for research, facilitating high-throughput screening of compounds and gene targets, and obtaining a variety of gene mutations Cells and use it to study cell phenotypes and pathogenic mechanisms, and there is no species difference with humans. Cells from patients are reprogrammed to obtain iPS, which can be used to study many genetic diseases. Of course, the acquisition of patient cells, the confirmation of pathogenic mutation genes, and the common analysis of cells with different genetic backgrounds all pose obstacles to the study of genetic diseases. However, these obstacles can be resolved by introducing disease-related ...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N5/10
CPCC12N5/0696C12N9/22C12N15/907C12N2510/00
Inventor 苏焕兴谭渊黄志健
Owner UNIVERSITY OF MACAU
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